Fusion proteins containing insulin-like growth factor-1 and epidermal growth factor and variants thereof and uses thereof

ABSTRACT

Fusion proteins comprising cytokines, particularly insulin-like growth factor-1 (IGF-1) and variants thereof, epidermal growth factor (EGF), and other ligands to the EGF receptor, are provided. The fusion proteins further comprise SEQ ID NO:1 or other segments having lysine, glutamic acid, or aspartic acid residues. Uses for the fusion proteins are also provided.

BACKGROUND

Currently 1.3 million people are diagnosed with cancer each year in theUnited States alone, and over 500,000 die. Treatment for most types ofcancers includes chemotherapy. Chemotherapy drugs are administeredsystemically and attack all cells of the body, particularly dividingcells, not just cancer cells. Thus, side effects from chemotherapy drugsare often severe. These include anemia, nausea, hair loss, and immunesuppression, including neutropenia, due to depletion of white bloodcells. The side effects often limit the dose of chemotherapy agents thatcan be administered.

Cancer cells are obligately glycolytic. That is, they must consumeglucose for their energy needs and they consume it anaerobically, whichyields less energy than aerobic respiration. As a consequence, cancercells must consume a large amount of glucose. Perhaps to assist withacquiring glucose, cancer cells from many types of cancer have beenobserved to have more insulin receptors than normal cells. (Ayre, S. G.,et al., 2000, Medical Hypotheses 55:330; Abita, J. F., et al., 1984,Leukemia Res. 8:213.) Recently, a method of cancer treatment termedinsulin potentiation therapy (IPT) that attempts to exploit the insulinreceptors of cancer cells has been introduced in the United States.(Ayre, S. G., et al., 2000, Medical Hypotheses 55:330.) The methodinvolves administering insulin to cancer patients, followed a short timelater by administering chemotherapy drugs. Lower doses of chemotherapydrugs are used, which reduces the side effects. It is purported that theinsulin somehow potentiates the effect of the chemotherapeutic agents onthe cancer cells, allowing the use of lower doses.

In vitro data is reported to show that when methotrexate is administeredwith insulin to breast cancer cells in tissue culture, the same percentcell killing is achieved with 10⁴ lower methotrexate concentrations thanwhen methotrexate is administered alone. (Alabaster, O., et al., 1981,Eur J. Cancer Clin. Oncol. 17:1223.) Methotrexate is a folic acidanalogue that leads to the depletion of tetrahydrofolate. Thisinterferes with thymidine and purine synthesis, and hence DNA synthesis.

Insulin does not greatly stimulate uptake of chemotherapeutic agents.One study has shown only a 2-fold stimulation of uptake of elipticine byMDA-MB-231 breast cancer cells when the cells were incubated withinsulin. (Oster, J. B., et al., 1981, Eur J. Cancer Clin. Oncol.17:1097.) Another study showed a 50% stimulation of uptake ofmethotrexate by breast cancer cells when the cells were incubated withinsulin. (Shilsky, R. L., et al., 1981, Biochem. Pharmacol. 30:1537.)Thus, the mechanism for insulin potentiation of methotrexatecytotoxicity must be primarily due to factors other than enhanceduptake.

Another receptor often found in greater numbers in cancer cells than innormal cells of the same tissue type is the insulin-type growth factor-1receptor (IGF-1 receptor or IGF-1R). IGF-1 is a peptide of 70 amino acidresidues having 40% identity with proinsulin. (Daughaday, W. H., et al.,1989, Endocrine Revs. 10:68.) Insulin and IGF-1 have somecross-reactivity with each other's receptor. (Soos, M. A., et al., 1993,Biochem. J. 290:419.) IGF-1 is secreted by the liver into thecirculatory system and stimulates growth of many cell types. IGF-1 isalso produced by many cell types throughout the body, including manycancers, for autocrine and paracrine effects. IGF-1 production isstimulated by growth hormone. (Stewart, C. H., et al., 1996, Physiol.Revs. 76:1005; Yakar, S., et al., 2002, Endocrine 19:239.)

To target the IGF receptor in cancer treatment, we have made compoundsfor treating cancer that have an anti-cancer chemotherapeutic agentlinked to an insulin-like growth factor-1 (IGF-1) receptor ligand (WO2005/041865; U.S. Pat. No. 8,501,906; US Published patent application20100197890, all incorporated by reference).

Another receptor often found overexpressed in cancer cells is theepidermal growth factor receptor (EGFR or ErbB-1).

SUMMARY

Improved ligands to the IGF-1R (type I IGF receptor) that areadvantageous for conjugating to chemotherapeutic agents are needed.Ligands to ErbB-1 that are advantageous for conjugation tochemotherapeutic agents are also needed. Improved expression inmicrobial hosts of cytokines is also desired.

We have synthesized a variant of IGF-1 we have named 765IGF having thesequence SEQ ID NO:2. SEQ ID NO:3 is the sequence of wild-type humanIGF-1. Residues 19-88 of SEQ ID NO:2 are identical to wild type IGF-1except that the Arginine at position 21 of SEQ ID NO:2 is a substitutionof glutamic acid at position 3 of wild-type IGF-1 (SEQ ID NO:3).Residues 19-88 of SEQ ID NO:2 correspond to R3-IGF, a variant form ofIGF-1 that has reduced binding for the soluble IGF-1 binding proteins(as compared to wild-type IGF-1). The soluble IGF-1 binding proteins aresoluble proteins in blood that bind circulating IGF-1 tightly. Whenbound to soluble IGF-1 binding proteins, IGF-1 is not available to bindto the membrane IGF receptor (type 1 IGF receptor, IGF-1R). In order tomore effectively target the IGF ligand portion of ourIGF-chemotherapeutic-agent conjugates to the IGF-1 receptor, we wantedthe ligand portion to be a variant having reduced binding affinity forthe soluble IGF-1 binding proteins.

Residues 1-18 of 765IGF (SEQ ID NO:2) are a leader sequence thatprovides a polyhistidine purification tag and several lysine residues.The lysine residues have amino groups that are available for conjugationto chemotherapeutic agents. So it is possible to couple morechemotherapeutic agent to 765IGF than to wild type IGF-1 or R3-IGF.

We have also conjugated (covalently attached) methotrexate (MTX) toamino groups of 765IGF to make a 765IGF-MTX conjugate, and have shownthat this conjugate inhibits growth of tumor cells in vitro.

765IGF has been found to have several surprising advantages:

-   -   The yield of purified 765IGF from a recombinant microbial host        per liter of fermented microbial host is higher than other        variants of IGF-1.    -   It binds at excellent affinity to the IGF receptor and displaces        more wild type IGF-1 than does another variant of IGF-1,        long-R3-IGF, suggesting that 765IGF may bind to a secondary site        on cancer cells that IGF-1 binds to.    -   765IGF conjugated to methotrexate yields a higher loading of        methotrexate (more methotrexate groups covalently attached per        IGF molecule) than two other IGF variants—IGF132 and        long-R3-IGF.    -   765IGF is more stable to storage than IGF132. IGF132 breaks down        to produce a significant amount of smaller molecular weight        fragments of IGF132. These smaller fragments are seen on        SDS-PAGE. 765IGF is more stable in storage and produces less of        these smaller fragments.

The leader sequence of 765IGF is SEQ ID NO:1. We have now used this sameleader sequence as an N-terminal leader on other cytokines and otherproteins expressed in E. coli, and in all cases tested so far it hasallowed purification of the protein in excellent yield in active form.

Thus, one embodiment of the invention provides a polypeptide comprisingSEQ ID NO:2.

Another embodiment provides a polypeptide comprising SEQ ID NO:1.

The amino terminal methionine of SEQ ID NO:1 and SEQ ID NO:2 issometimes cleaved off of the polypeptide in E. coli, so one embodimentprovides a polypeptide comprising residues 2-18 of SEQ ID NO:1.

Another embodiment provides a polypeptide comprising (a) SEQ ID NO:1 orresidues 2-18 of SEQ ID NO:1 and (b) SEQ ID NO:3, SEQ ID NO:4, residues2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 ofSEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or residues 32-111 of SEQID NO:13, or a variant 90% or more identical to SEQ ID NO:3, SEQ IDNO:4, residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10,residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or toresidues 32-111 of SEQ ID NO:13.

Another embodiment provides a compound comprising an anti-cancerchemotherapeutic agent covalently attached to a polypeptide comprising(a) SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 and (b) SEQ ID NO:3, SEQID NO:4, residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10,residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, orresidues 32-111 of SEQ ID NO:13, or a variant 90% or more identical toSEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9, residues 40-89of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 ofSEQ ID NO:12, or to residues 32-111 of SEQ ID NO:13. Preferably theanti-cancer chemotherapeutic agent is covalently attached to lysine sidechains of SEQ ID NO:1 in the polypeptide.

Another embodiment provides a method of inhibiting the growth of cancercells comprising contacting the cancer cells with the compoundcomprising an anticancer chemotherapeutic agent covalently attached to apolypeptide comprising (a) SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1and (b) SEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9, residues40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148of SEQ ID NO:12, or residues 32-111 of SEQ ID NO:13, or a variant 90% ormore identical to SEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ IDNO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, or to residues 32-111 of SEQ ID NO:13.

Likewise, another embodiment provides a method of treating cancer in amammal comprising administering to the mammal the same compound.

It is believed that stimulating cancer cells to divide with IGF-1 atapproximately the same time that radiation is administered (i.e, withinapproximately 6 hours before or after administration of the radiation)sensitizes the cancer cells to be killed by the radiation. (See US20060258589.) Thus, one embodiment provides a method of treating cancerin a mammal comprising: administering a polypeptide comprising SEQ IDNO:2 or residues 2-88 of SEQ ID NO:2 to the mammal and administeringradiation to the mammal.

Likewise, it is believed that stimulating cancer cells to divide withIGF-1 at approximately the same time that chemotherapy is administered(i.e, within approximately 6 hours before or after administration of thechemotherapy) sensitizes the cancer cells to be killed by thechemotherapy. (See U.S. Pat. No. 8,501,906.) Thus, one embodimentprovides a method of treating cancer in a mammal comprising:administering to the mammal an anti-cancer chemotherapeutic agent and apolypeptide comprising SEQ ID NO:2 or residues 2-88 of SEQ ID NO:2.

Another embodiment provides a fusion polypeptide comprising: (a) SEQ IDNO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ IDNO:12, or residues 32-111 of SEQ ID NO:13, or a variant 90% or moreidentical to SEQ ID NO:3, SEQ ID NO:4; residues 2-54 of SEQ ID NO:9,residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, or to residues 32-111 of SEQ ID NO:13;and (b) a polypeptide segment or segments N-terminal to (a) orC-terminal to (a) or both N-terminal to (a) and C-terminal to (a);wherein polypeptide segment or segments (b) total 3-40 amino acidresidues and comprise 3-20 amino acid residues that are lysine residuesor 3-20 amino acid residues that are aspartic acid or glutamic acidresidues.

Another embodiment provides a compound comprising an anti-cancerchemotherapeutic agent covalently attached to the fusion polypeptidecomprising: (a) SEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9,residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, or residues 32-111 of SEQ ID NO:13, ora variant 90% or more identical to SEQ ID NO:3, SEQ ID NO:4; residues2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 ofSEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or to residues 32-111 ofSEQ ID NO:13; and (b) a polypeptide segment or segments N-terminal to(a) or C-terminal to (a) or both N-terminal to (a) and C-terminal to(a); wherein polypeptide segment or segments (b) total 3-40 (or 3-30, or3-20) amino acid residues and comprise 3-20 amino acid residues that arelysine residues or 3-20 amino acid residues that are aspartic acid orglutamic acid residues. Preferably, the chemotherapeutic agent iscovalently attached to the lysine residues or aspartic acid or glutamicacid residues of segment or segments (b) of the fusion polypeptide.

Another embodiment provides a method of inhibiting the growth of cancercells comprising contacting the cancer cells with the compoundcomprising an anti-cancer chemotherapeutic agent covalently attached tothe fusion polypeptide comprising: (a) SEQ ID NO:3, SEQ ID NO:4,residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or residues32-111 of SEQ ID NO:13, or a variant 90% or more identical to SEQ IDNO:3, SEQ ID NO:4; residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ IDNO:12, or to residues 32-111 of SEQ ID NO:13; and (b) a polypeptidesegment or segments N-terminal to (a) or C-terminal to (a) or bothN-terminal to (a) and C-terminal to (a); wherein polypeptide segment orsegments (b) total 3-40 amino acid residues and comprise 3-20 amino acidresidues that are lysine residues or 3-20 amino acid residues that areaspartic acid or glutamic acid residues. Preferably, thechemotherapeutic agent is covalently attached to the lysine residues oraspartic acid or glutamic acid residues of segment or segments (b) ofthe fusion polypeptide.

Likewise, another embodiment provides a method of treating cancer in amammal comprising administering to the mammal the same compound.

It is shown herein that a conjugate of bendamustine to a fusion proteincomprising the soluble form of epidermal growth factor in a fusionprotein with the leader sequence of SEQ ID NO:1 is effective to treatand in some cases cure cancer in a mouse in vivo model with a xenograftof a cancer cell line high in ErbB-1 (EGF) receptors. The bendamustineconjugate inhibited cell line growth of the same cell line at more than1000-fold lower concentration than free bendamustine. Thus, oneembodiment provides a compound comprising bendamustine covalentlyattached to a cytokine that is a ligand to ErbB-1. The cytokine may bepart of a fusion protein, but is not necessarily part of a fusionprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an IGF receptor binding assay showing % ofradioactive signal bound (radioactive IGF-1) versus concentration of765IGF or long-R3-IGF.

FIG. 2 shows a plot of MCF7 cell growth inhibition by 765IGF-MTX used todetermine an IC₅₀ of 765IGF-MTX for growth inhibition.

FIG. 3 shows the results of an assay for inhibition of dihydrofolatereductase (DHFR) by 765IGF-MTX.

FIG. 4 shows a matrix-assisted laser desorption time of flight(MALDI-TOF) mass spectrum of 765EGF-bendamustine.

FIG. 5 shows a plot of in vitro proliferation inhibition with A-431cells by both 765EGF-bendamustine and free bendamustine.

FIG. 6 is a plot of in vitro proliferation inhibition by765EGF-bendamustine of A-431 cells on which the IC₅₀ of765EGF-bendamustine is calculated.

FIG. 7 is a plot of xenograft tumor growth versus time after treatmentfor the various treatment groups in mice with A-431 xenografts andreceiving 765EGF-bendamustine treatment.

FIG. 8 is a plot of xenograft tumor growth in the 765EGF-bendamustinestudy with pooled data for the 0 and 50 pEq/g groups, the 200 or morepE/g groups, and the IV 200 pEq/g group.

DETAILED DESCRIPTION Definitions

The term “anti-cancer chemotherapeutic agent” refers to a synthetic,biological, or semi-synthetic compound that is not an enzyme and thatkills cancer cells or inhibits the growth of cancer cells while havingless effect on non-cancerous cells.

The term “treating cancer” includes, e.g., preventing metastasis,inhibiting growth of a cancer, stopping the growth of cancer, or killingcells of a cancer.

The term “binding affinity” of a ligand for a particular receptor refersto the association constant K_(A) (the inverse of the dissociationconstant K_(D)) or to experimentally determined approximations thereof.

The term “anti-metabolite” refers to an anti-cancer chemotherapeuticagent that bears a structural similarity to a naturally occurringsubstance, interacts with enzymes as an inhibitor or a substrate, andinterferes with cellular processes. Examples include methotrexate,fluorouracil, floxuridine, fludarabine, mercaptopurine, thioguanine,cytarabine, azacytidine, cladribine, and pentostatin.

The term “cytokine” refers to nonantibody proteins released by one cellpopulation on contact with specific antigen, which act as intercellularmediators, as in the generation of an immune response. Cytokinesinclude, for example, insulin, insulin-like growth factor 1 (IGF-1),epidermal growth factor, transforming growth factor alpha, transforminggrowth factor beta, and interleukins.

The “IGF-1 receptor” is also known in the literature as the type 1 IGFreceptor.

“Containing” as used herein is open-ended; i.e, it allows the inclusionof other unnamed elements and has the same meaning as “comprising.”

The “EGF receptor” as used herein, refers to ErbB-1.

Description

We have expressed in E. coli, from a recombinant vector with expressioncontrolled by a T7 promoter and induced with IPTG, a fusion proteinhaving the sequence of SEQ ID NO:2. This protein has the leader sequenceat its N-terminus of SEQ ID NO:1, which provides a polyhis tag forpurification and several additional lysine residues. The C-terminal ofthe protein is residues 19-88 and corresponds to R3-IGF, which is humanwild type IGF-1 sequence with an arginine at position 21 of SEQ ID NO:2that replaces the native glutamic acid at position 3 of wild-type IGF-1(SEQ ID NO:3).

R3-IGF is a variant IGF-1, as discussed below.

765IGF (SEQ ID NO:2) comprising SEQ ID NO:1 as a leader sequencefollowed by R3-IGF expressed at a high yield and purified at a higheryield than other IGF fusion protein constructs comprising differentleader sequences. It was more stable to storage than IGF132, anothervariant of IGF-1. It also refolded with almost 100% yield of activeform, and it displaced more wild-type IGF-1 from its receptor on MCF7cells than did long-R3-IGF, another variant of IGF-1.

The SEQ ID NO:1 leader also provides five lysine residues. A765IGF-methotrexate conjugate was prepared by covalently attachingmethotrexate through one of its carboxyl groups by amide bond to aminogroups on 765IGF. 765IGF has nine amino groups, including eight lysineside chains (five of these in the SEQ ID NO:1 leader) and the aminoterminal alpha-amino group. The 765IGF-MTX had an average of about 8methotrexate groups attached per IGF monomer. Conjugates to longR3-IGFand IGF132 had fewer methotrexate groups per IGF monomer. So this wasanother advantage of the SEQ ID NO:1 leader.

A fusion protein called 765EGF with the SEQ ID NO:1 leader at theN-terminus fused to the sequence of mature soluble form EGF was alsosynthesized (SEQ ID NO:8). This also expressed from a recombinant vectorunder the control of a T7 promoter in E. coli to high yield and purifiedto good yield. It also refolded to a biologically active form.

The SEQ ID NO:1 leader is thus generally applicable for expressingproteins in good yield from microbial hosts, particularly E. coli, andfor efficient purification in good yield. It is particularly applicablefor expression of cytokines.

R3-IGF is a variant IGF-1 in a fusion protein with SEQ ID NO:1 in SEQ IDNO:2. It is a variant that activates the IGF receptor (IGF-1R) but hasreduced binding affinity for the soluble IGF binding proteins (ascompared to wild-type IGF-1) (Francis, G. L., et al. 1992, J. Mol.Endocrinol. 8:213-223; Tomas, F. M. et al., 1993, J. Endocrinol.137:413-421). Soluble IGF binding proteins are natural serum proteinsthat bind to IGF-1, holding it in circulation and extending itsbiological half-life. But when IGF-1 is bound to the IGF bindingproteins it cannot bind to the membrane IGF receptor (IGF-1R). (Clemons,D. R., 1998, Mol. Cell. Endocrinol. 140:19-24.) For that reason,variants of IGF-1 that have reduced binding to the soluble IGF bindingproteins are more active in vivo than wild-type IGF-1 and more rapidlytarget the IGF receptor.

Binding affinity for IGF binding proteins can be tested with ratL6-myoblast-conditioned medium. The medium from growth of rat L6myoblasts (0.2 ml) is mixed with 8,000 cpm ¹²⁵I-IGF-1 (approximately0.05 uCi) in 0.3 ml final volume of 50 mM sodium phosphate, pH 6.5,0.25% bovine albumin and test competitor (wild type IGF-1 or an IGFvariant) at 0.1 nM to 1 uM final concentration. After incubation 90minutes at room temperature, to separate bound and free tracer an icecold rapidly stirred suspension of charcoal at 5 mg/ml in assay buffercontaining 0.2 mg/ml protamine sulfate is added to the sample, and after8 minutes on ice, the mixture is centrifuged 20 minutes at 5,000×g.Radioactivity in the supernatant is counted in a gamma counter. Thebinding affinity of a variant can be compared to that of wild-type IGFto determine whether a variant has reduced binding affinity for thesoluble IGF binding proteins.

Thus, in some embodiments, the polypeptides described herein compriseSEQ ID NO:1 and a variant IGF-1 that has reduced binding affinity forthe soluble IGF binding proteins.

Some specific variants of IGF-1 with reduced binding affinity to thesoluble IGF binding proteins include IGF132 (SEQ ID NO:4) (disclosed inU.S. Pat. No. 4,876,242), LONG-R3-IGF (SEQ ID NO:5), R3-IGF (SEQ IDNO:6), and des(1-3)IGF1 (SEQ ID NO:7), which lacks the first threeresidues of wild-type IGF-1. (LongR3-IGF, R3-IGF, and des(1-3)IGF1, aredescribed in Francis, G. L., et al. 1992, J. Mol. Endocrinol. 8:213-223;Tomas, F. M. et al., 1993, J. Endocrinol. 137:413-421). Thus, inparticular embodiments, the polypeptide that is a variant IGF-1 withreduced binding to the soluble IGF-1 binding proteins comprises any oneof SEQ ID NOS:4-7.

The IGF receptor may be targeted in cancer with conjugates comprising(a) an anti-cancer chemotherapeutic agent covalently coupled to (b) anIGF receptor ligand such as IGF-1 or the IGF variants described hereinin a polypeptide fusion comprising SEQ ID NO:1 or residues 2-18 of SEQID NO:1

Preferably, the IGF-1 receptor ligand with reduced affinity for solubleIGF-1 binding proteins has at least 5-fold, more preferably at least10-fold, more preferably still at least 100-fold lower binding affinityfor soluble IGF-1 binding proteins than wild-type IGF-1. Bindingaffinity for the soluble IGF-1 binding proteins can be measured by acompetition binding assay against labeled IGF-1 (e.g., ¹²⁵I IGF-1),using a mixture of purified IGF-1 binding proteins or rat L6myoblast-conditioned medium (a naturally produced mixture of IGF-1binding proteins), as described in Francis, G. L., et al. (1992, J. Mol.Endocrinol. 8:213-223); Szabo, L. et al. (1988, Biochem. Biophys. Res.Commun. 151:207-214); and Martin, J. L. et al. (1986, J. Biol. Chem.261:8754-8760). Preferably, the variant IGF-1 has an IC₅₀ in acompetition binding assay against labeled wild-type IGF-1 for binding tosoluble IGF-1 binding proteins in L6 myoblast-conditioned medium ofgreater than 10 nM, more preferably greater than 100 nM.

Preferably, the variant IGF-1 with reduced affinity for soluble IGF-1binding proteins has affinity for the IGF-1 receptor that is close towild-type IGF-1 (e.g., less than 30-fold greater than wild-type IGF-1,more preferably less than 10-fold greater than wild-type IGF-1). Inspecific embodiments, the variant IGF-1 has an IC₅₀ in a competitionbinding assay against labeled wild-type IGF-1 for binding to IGF-1receptors (e.g., on MCF-7 cells) of less than 50 nM, more preferablyless than 10 nM, more preferably still less than 5 nM, more preferablystill less than 3 nM). This assay is described in Ross, M. et al. (1989,Biochem. J. 258:267-272) and Francis, G. L., et al. (1992, J. Mol.Endocrinol. 8:213-223), and in Example 4 herein.

Another receptor often found in greater numbers in cancer cells than innormal cells of the same tissue type is the epidermal growth factor(EGF) receptor. (Nicholson, R. I. et al., 2001, Eur. J. Cancer37:S9-S15. Kopp, R., et al., 2003, Recent Results in Cancer Research162:115-132. Fox, S. B. et al., 1994, Breast Cancer Res. Treat.29:41-49.) The EGF receptor, also known as ErbB-1, is activated byseveral agonists, including EGF itself, transforming growth factor alpha(TGFα), amphiregulin (AR), heparin-binding EGF-like growth factor(HB-EGF), and betacellulin (BTC). (Beerli, R. R. et al., 1996, J. Biol.Chem. 271:6071-6076. Earp, H. S., et al., 2003, Trans. Am. Clin. Clim.Assoc. 114:315-333.) Three other receptors are also considered membersof the EGF family of receptors. They are ErbB-2, ErbB-3, and ErbB-4(also known as HER2, HER3, and HER4, for human EGF receptor 2, 3, and 4,respectively). These receptors, especially ErbB-2, are also oftenoverexpressed on cancerous cells. The receptors ErbB-2 and ErbB-4 aretyrosine kinases. The EGF receptor agonists listed above bind moststrongly to the EGF receptor. They bind less tightly to the otherreceptors in the EGF receptor family. Neu differentiation factors(NDFs)/heregulins are ligands for EbrB-3 and ErbB-4. (Beerli, R. R.,1996, J. Biol. Chem. 271:6071-6076. Carraway, K. L. et al., 1994, J.Biol. Chem. 269:14303-14306. Plowman, G. D., et al., 1993, Nature366:473-475.)

Thus, EGF, TGFα, amphiregulin, HB-EGF, BTC, and NDFs are alsopolypeptides that may be in fusion proteins with SEQ ID NO:1.

The sequence of a precursor of EGF is SEQ ID NO:9. In mature EGF, theamino terminal methionine of SEQ ID NO:9 is removed. (Gregory, H., 1975,Nature 257:325-327.) The sequence of the precursor of TGFα is SEQ IDNO:10. Mature TGFα is thought to be residues 40-89 of SEQ ID NO:10.(Qian, J. F., et al., 1993, Gene 132:291-296. Higashayaam, S., et al.,1991, Science 251:936-939.) The sequence of the precursor ofamphiregulin is SEQ ID NO:11. Mature amphiregulin is thought to beresidues 101-184 of SEQ ID NO:11. (Plowman, G. D., et al., 1990, Mol.Cell. Biol. 10:1969-1981.) The sequence of the precursor of HB-EGF isSEQ ID NO:12. Mature HB-EGF is thought to be residues 63-148 of SEQ IDNO:12. (Higashayama, S. et al., 1992, J. Biol. Chem. 267:6205-6212.Higashayaam, S., et al., 1991, Science 251:936-939.) The sequence of theprecursor of betacellulin is SEQ ID NO:13. Mature betacellulin isthought to be residues 32-111 of SEQ ID NO:13. (Sasada, R. et al., 1993,Biochem. Biophys. Res. Comm. 190:1173-1179.) Cysteine residues 7 with21, 15 with 32, and 34 with 43 of SEQ ID NO:9 form disulfide bridges toeach other in mature EGF. (Gregory, H., 1975, Nature 257:325-327.) Thehomologous cysteine residues in the other natural EGF receptor ligandsalso form disulfide bridges. (Higashayaam, S., et al., 1991, Science251:936-939.) Another polypeptide ligand to the EGF receptor is achimera of sequences from natural EGF receptor ligands, e.g., thechimera E4T, which is a chimera of EGF and TGFα sequences, and is a moreactive agonist than either EGF or TGFα. (Lenferink, A. E. G., et al.,1998, EMBO J. 17:3385-3397. Kramer, R. H., et al., 1994, J. Biol. Chem.269:8708-8711.) Active chimeras that are agonists of ErbB-1 such as E4Tmay also be in fusion proteins with SEQ ID NO:1.

The fusion polypeptides comprising SEQ ID NO:1 or residues 2-18 of SEQID NO:1 and IGF-1 or a variant IGF-1 as described herein can also beused to enhance the effectiveness of chemotherapy or radiation by beingadministered to a cancer patient within 6 hours of administration of achemotherapy agent or radiation therapy to the patient, as is describedin WO 2005/041865 and U.S. Pat. No. 8,501,906 and U.S. PatentApplication publication No. 20060258589.

Another embodiment of the invention provides a fusion protein comprising(a) IGF or an IGF variant or EGF or another ErbB-1 ligand or a variantthereof and (b) another polypeptide segment that provides additionalamino acid residues to which a chemotherapeutic agent may be conjugated,particularly lysine, glutamic acid residues, or aspartic acid residues.IGF-1 has only 3 lysine residues and EGF has only 2 lysine residues. Inorder to have a higher loading of chemotherapeutic agent, we have foundit is advantageous to have a fusion protein segment added to the IGF-1or EGF segment (a) that has additional reactive amino acid residues,particularly lysine residues, to which a chemotherapeutic agent can beconjugated.

Thus, one embodiment of the invention provides a fusion polypeptidecomprising: (a) SEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9,residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, or residues 32-111 of SEQ ID NO:13, ora variant 90% or more identical to SEQ ID NO:3, SEQ ID NO:4, residues2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 ofSEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or to residues 32-111 ofSEQ ID NO:13; and (b) a polypeptide segment or segments N-terminal to(a) or C-terminal to (a) or both N-terminal to (a) and C-terminal to(a); wherein polypeptide segment or segments (b) total 3-40 (or 3-20 or3-60 or 5-20 or 5-60) amino acid residues and comprise 3-20 amino acidresidues that are lysine residues or 3-20 amino acid residues that areaspartic acid or glutamic acid residues.

In particular embodiments, the polypeptide segment or segments (b)comprises 3-20 (or 3-10 or 5-20 or 5-10) amino acid residues that arelysine. In a specific embodiment, at least 20% of the residues ofsegment or segments (b) are lysine residues.

In other embodiments, the polypeptide segment or segments (b) comprise3-20 (or 3-10, or 5-10, or 5-10) amino acid residues that are asparticacid or glutamic acid residues (i.e., the total of aspartic and glutamicacid residues equals the cited number). In a specific embodiment, atleast 20% of the residues of segment or segments (b) are aspartic acidor glutamic acid residues.

In a specific embodiment of this fusion protein the polypeptide segment(a) comprises IGF-1 or a variant of IGF-1 at least 90% identical to anyone of SEQ ID NOS:3 and 4.

In another specific embodiment of this fusion protein, the polypeptidesegment (a) comprises residues 2-54 of SEQ ID NO:9, residues 40-89 ofSEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQID NO:12, or residues 32-111 of SEQ ID NO:13, or a variant 90% or moreidentical to residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ IDNO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ IDNO:12, or to residues 32-111 of SEQ ID NO:13.

Another embodiment of the invention provides a compound comprising achemotherapeutic agent covalently attached to the fusion polypeptidecomprising a ligand to ErbB-1 or IGFR1.

In a more specific embodiment, the chemotherapeutic agent is covalentlyattached to one or more lysine residue side chains of segment orsegments (b) in the polypeptide.

In a more specific embodiment where the chemotherapeutic agent iscovalently attached to one or more lysine residue side chains of segmentor segments (b), the chemotherapeutic agent may be one with a freecarboxyl group, such as methotrexate, chlorambucil, or bendamustine.

In specific embodiments of the methods described herein, the cancertreated is lung cancer, prostate cancer, colorectal cancer, breastcancer, pancreatic cancer, leukemia, liver cancer, stomach cancer,ovarian cancer, uterine cancer, testicular cancer, brain cancer,non-Hodgkin's lymphoma, Hodgkin's lymphoma, Ewing's sarcoma,osteosarcoma, neuroblastoma, rhabdomyosarcoma, melanoma, or braincancer.

In specific embodiments of the methods with the conjugates comprisingfusion proteins comprising a cytokine that is a ligand to ErbB-1, thecancer is an epithelial cell cancer.

In particular embodiments, the chemotherapeutic agent conjugated to thepolypeptide is mechlorethamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, thiotepa, hexamethylmelamine, busulfan, carmustine,lomustine, semustine, streptozocin, decarbazine, vincristine,vinblastine, etoposide, teniposide, paclitaxel, docetaxel, daunorubicin,idarubicin, doxorubicin, epirubicin, dactinomycin, plicamycin, mitomycinC, bleomycin, mitoxantrone, methotrexate, fluorouracil, floxuridine,fludarabine, mercaptopurine, thioguanine, cytarabine, azacytidine,cladribine, pentostatin, cisplatin, carboplatin, mitotane, procarbazine,or amsacrine.

Guidelines for Coupling Anti-Cancer Chemotherapeutic Agents to ReceptorLigands

The natural ligands to the insulin and IGF-1 receptors are proteins,namely insulin, IGF-1, and IGF-2. Chemotherapeutic agents are typicallycoupled to proteins through the reactive groups present on proteins.These include the N-terminal alpha-amino group, the C-terminalalpha-carboxyl group, the side-chain amino group of lysine, theside-chain carboxyl groups of aspartic acid and glutamic acid, the sidechain thiol of cysteine, and the side chain of arginine. Other reactiveside chains found on proteins are the side-chain hydroxyl of serine andthreonine, the hydroxyaryl of tyrosine, the imidazole of histidine, andthe methionine side chain.

Many of the same reactive groups are found on chemotherapeutic agentsand on non-proteinaceous ligands of the insulin and IGF-1 receptors.Thus, many of the principles of modification and cross-linking ofproteins discussed herein also apply to modification and cross-linkingof chemotherapeutic agents and non-proteinaceous ligands.

The chemistry and principles of protein conjugation and cross-linkingare described in Wong, Shan S., Chemistry of Protein Conjugation andCross-Linking, 1991, CRC Press, Boca Raton, Fla. Other sources forinformation on this chemistry include the Pierce Biochemistry catalog;and Greene, T. W., and Wutz, P. G. M., Protecting Groups in OrganicSynthesis, second edition 1991, John Wiley & Sons, Inc., New York, andreferences cited therein.

The strongest nucleophile of amino acid side chains is the thiol ofreduced cysteine side chains. The thiol reacts with most proteinmodifying reagents. Alpha-haloacetamides and maleimides are consideredto react specifically with cysteine residues, particularly at pH 7.0 andbelow. Thiols also react by disulfide interchange with disulfidereagents.

Amino groups are the next-strongest nucleophiles found on proteins.Aldehydes react with amino groups to form Schiff bases. The Schiff basesare hydrolyzable, which can be an advantage in the present invention.With uptake into cancer cells of a ligand-chemotherapeutic agentconjugate, in some cases it is necessary that the chemotherapeutic agentis cleaved from the conjugate for it to be active. This is betteraccomplished if the chemotherapeutic agent is linked to the ligand by acleavable linkage, such as a hydrolyzable linkage. Cleavable linkagescan be cleaved spontaneously or by enzymes in the cell. For instance,amide bonds are cleaved by certain enzymes, including proteases. ASchiff base linkage spontaneously hydrolyzes at an appreciable rate. Adisulfide linkage is expected to be reductively cleaved in theintracellular reducing environment of a cancer cell.

The Schiff base formed by reaction of an amino group with an aldehydecan be stabilized by reduction with, for instance, sodium borohydride orpyridine borane. Pyridine borane has the advantage of not reducingdisulfides, which are found in insulin, IGF-1, and IGF-2 and areessential for the structure of those proteins.

Sugars or other moieties having hydroxyl groups on adjacent carbons,which are found in some chemotherapeutic agents, can be modified toreact with amino groups by oxidizing the sugars with, for instance,periodate. This cleaves between the carbons and produces a dialdehyde.The aldehyde groups will react with amino groups.

A dialdehyde, such as glutaraldehyde, will cross-link two moleculeshaving amino groups.

Other amino reagents include activated carbonyls, such asN-hydroxysuccinimide esters, p-nitrophenyl esters, or acid anhydrides(e.g., succinic anhydride).

Amino groups also react with sulfonyl halides and aryl halides (e.g,2,4-dinitrofluorobenzene).

Amino groups also react with isocyanates and isothiocyanates to formurea or thiourea derivatives.

Imidoesters are the most specific acylating agents for amino groups.Imidoesters react specifically with amines to from imidoamides at pHsbetween about 7 and 10. This reaction has the advantage of maintainingcharge stability by generating a positively charged group, theimidoamide, at the former amino group. Imidoamides also slowly hydrolyzeat pHs above neutrality, which can also be an advantage in that thehydrolysis can release free chemotherapeutic agent in the cancer cell.

Carboxyl groups react specifically with diazoacetate and diazoacetamideunder mild acid conditions, e.g., pH 5.

The most important chemical modification of carboxyls usescarbodiimides, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (CMC) and3-(3-dimethylaminopropyl)carbodiimide (EDC). In the presence of anamine, carbodiimides form an amide bond to the carboxyl in two steps. Inthe first step, the carboxyl group adds to the carbodiimide to form anO-acylisourea intermediate. Subsequent reaction with an amine yields thecorresponding amide.

A particularly important carbodiimide reaction is its use in activatingcarboxyls with N-hydroxysuccinimide to form an N-hydroxysuccinimideester.

Arginine reacts with vicinal dialdehydes or diketones, such as glyoxal,2,3-butanedione, and 1,2-cyclohexanedione. Borate may stabilize theadduct, if stabilization is desired.

The reactive groups can also be interchanged with other reactive groupsby some of the above reactions. For instance, modification of an aminogroup with an acid anhydride such as succinic anhydride, replaces thepositively charged amino group with a free carboxyl group. Likewise,reaction of a carboxyl group with a carbodiimide and a diamine, such asethylene diamine, replaces the carboxyl group with a free amino group.

Cross-Linking:

Reagents containing two of the reactive groups described above, forinstance two amino-reactive groups or an amino-reactive and athiol-reactive group, can be used to cross-link a chemotherapeutic agentcontaining one of the appropriate groups to an insulin or IGF-1 receptorligand containing the other appropriate group. In addition, a carboxyl(of, e.g., a chemotherapeutic agent) activated with a carbodiimide or acarbodiimide and N-hydroxysuccinimide can react with an amino group (of,e.g., a protein ligand) to form an amide bond cross-link.

The activated carboxyl is stable enough to be isolated, but will thenreadily react with amino groups to form an amide bond.

Succinimides such as N-succinimidyl-3-[2-pyridyldithio]propionate (SPDP)can be used to couple two compounds through amino groups. (See PierceBiotechnology catalog, and Thorpe, P. E. et al. 1982, Immunol. Rev.62:119-158.)

Statements of Invention:

1. A polypeptide comprising SEQ ID NO:1 or comprising residues 2-18 ofSEQ ID NO:1.

2. The polypeptide of statement 1 wherein the polypeptide has anN-terminus and SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 is at theN-terminus of the polypeptide.

3. The polypeptide of any of statements 1-2 wherein the polypeptide is afusion protein further comprising a cytokine.

4. The polypeptide of statement 3 wherein the cytokine is a ligand toErbB-1 or IGFR1.

4b. The polypeptide of statement 3 wherein the cytokine is tumornecrosis factor-alpha.

4c. The polypeptide of statement 4b wherein the polypeptide comprisesSEQ ID NO:17 or residues 2-175 of SEQ ID NO:17.

5. The polypeptide of statement 4 wherein the polypeptide comprises (a)SEQ ID NO:1 or residues 2-18 of SEQ ID NO:1 and (b) SEQ ID NO:3, SEQ IDNO:4, residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10,residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, orresidues 32-111 of SEQ ID NO:13, or a variant 90% or more identical toSEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9, residues 40-89of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 ofSEQ ID NO:12, or to residues 32-111 of SEQ ID NO:13.5a. The polypeptide of statement 5 wherein segment (b) is SEQ ID NO:3,SEQ ID NO:4, or a variant 90% or more identical to SEQ ID NO:3 or to SEQID NO:4.5b. The polypeptide of statement 5 wherein segment (b) is residues 2-54of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQID NO:11, residues 63-148 of SEQ ID NO:12, or residues 32-111 of SEQ IDNO:13, or a variant 90% or more identical to residues 2-54 of SEQ IDNO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, or to residues 32-111 of SEQ ID NO:13.5c. The polypeptide of statement 5b wherein segment (b) is residues 2-54of SEQ ID NO:9.5d. The polypeptide of statement 5 wherein segment (b) is a variant of90% or more identical to SEQ ID NO:3 selected from SEQ ID NO:6 and SEQID NO:7.6. The polypeptide of statement 5 wherein the polypeptide comprises SEQID NO:2 or residues 2-88 of SEQ ID NO:2.7. A compound comprising an anti-cancer chemotherapeutic agentcovalently attached to the polypeptide of statement 5.8. The compound of statement 7 wherein the chemotherapeutic agent iscovalently attached to one or more lysine residue side chains ofresidues 2-18 of SEQ ID NO:1 in the polypeptide.9. The compound of statement 7 wherein the chemotherapeutic agent ismethotrexate.10. The compound of statement 5 wherein the chemotherapeutic agent isselected from the group consisting of methotrexate, chlorambucil, andbendamustine.11. The compound of statement 9 comprising methotrexate covalentlyattached to a polypeptide comprising SEQ ID NO:2 or residues 2-88 of SEQID NO:2.12. The compound of statement 11 wherein the methotrexate is attached byamide bonds between carboxyl groups of the chemotherapeutic agent andamino groups of the polypeptide.13. A method of inhibiting the growth of cancer cells comprisingcontacting the cancer cells with a compound of any one of statements7-12.14. The method of statement 13 wherein the contacting is in vitro.15. The method of statement 13 wherein the contacting is in vivo.16. The method of statement 15 wherein the contacting is in vivo in ahuman.17. The method of statement 15 wherein the contacting is in vivo in anonhuman mammal.18. A method of treating cancer in a mammal comprising: administering tothe mammal a compound of any one of statements 7-12.19. The method of statement 18 wherein the mammal is not a human.20. The method of statement 18 wherein the mammal is a human.21. A method of treating cancer in a mammal comprising:

administering a polypeptide comprising SEQ ID NO:2 or residues 2-88 ofSEQ ID NO:2 to the mammal and administering radiation to the mammal.

22. A method of treating cancer in a mammal, comprising:

administering to the mammal an anti-cancer chemotherapeutic agent and apolypeptide comprising SEQ ID NO:2 or residues 2-88 of SEQ ID NO:2.

23. A fusion polypeptide comprising:

(a) SEQ ID NO:3, SEQ ID NO:4, residues 2-54 of SEQ ID NO:9, residues40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148of SEQ ID NO:12, or residues 32-111 of SEQ ID NO:13, or a variant 90% ormore identical to SEQ ID NO:3, SEQ ID NO:4; residues 2-54 of SEQ IDNO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, or to residues 32-111 of SEQ ID NO:13;and

(b) a polypeptide segment or segments N-terminal to (a) or C-terminal to(a) or both N-terminal to (a) and C-terminal to (a);

wherein polypeptide segment or segments (b) total 3-40 amino acidresidues and comprise 3-20 amino acid residues that are lysine residuesor 3-20 amino acid residues that are aspartic acid or glutamic acidresidues.

23a. The fusion polypeptide of statement 23 wherein segment (b) is SEQID NO:1 or residues 2-18 of SEQ ID NO:1 and is N-terminal to segment(a).

23b. The fusion polypeptide of statement 23 wherein polypeptide segmentor segments (b) comprise 3-10 amino acid residues that are lysineresidues or 3-10 amino acid residues that are aspartic acid or glutamicacid residues.

24. The fusion polypeptide of statement 23 wherein the polypeptidesegment (b) comprises 3-20 amino acid residues that are lysine.

25. The fusion protein of statement 23 or 24 wherein the polypeptidesegment (a) comprises IGF-1 or a variant of IGF-1 at least 90% identicalto any one of SEQ ID NOS:3 and 4.

26. The fusion protein of statement 23 or 24 wherein the polypeptidesegment (a) comprises residues 2-54 of SEQ ID NO:9, residues 40-89 ofSEQ ID NO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQID NO:12, residues 32-111 or SEQ ID NO:13, or a variant 90% or moreidentical to residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ IDNO:10, residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ IDNO:12, or to residues 32-111 of SEQ ID NO:13.27. A compound comprising a chemotherapeutic agent covalently attachedto the fusion polypeptide of any one of statements 23-26.27a. The compound of statement 27 wherein the polypeptide segment (a)comprises SEQ ID NO:3 or SEQ ID NO:4 or a variant at least 90% identicalto any one of SEQ ID NOS:3 and 4.27b. The compound of statement 27a wherein the fusion polypeptidecomprises SEQ ID NO:2 or residues 2-88 of SEQ ID NO:2.27c. The compound of statement 27 wherein the polypeptide segment (a)comprises residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10,residues 101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12,residues 32-111 or SEQ ID NO:13, or a variant 90% or more identical toresidues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or to residues32-111 of SEQ ID NO:13.28. The compound of statement 27 wherein the chemotherapeutic agent iscovalently attached to one or more lysine residue side chains of segment(b) in the polypeptide.29. The compound of statement 28 wherein the chemotherapeutic agent ismethotrexate.30. The compound of statement 28 wherein the chemotherapeutic agent isselected from the group consisting of methotrexate, chlorambucil, andbendamustine.31. The compound of statement 27 wherein the chemotherapeutic agent iscovalently attached to one or more aspartic acid or glutamic acid sidechains of segment (b) in the polypeptide.32. A method of inhibiting the growth of cancer cells comprisingcontacting the cancer cells with a compound of any one of statements27-31.33. The method of statement 32 wherein the contacting is in vitro.34. The method of statement 32 wherein the contacting is in vivo.35. The method of statement 34 wherein the contacting is in vivo in ahuman.36. The method of statement 34 wherein the contacting is in vivo in anonhuman mammal.37. A method of treating cancer in a mammal comprising:

administering to the mammal a compound of any one of statements 27-31.

38. The method of statement 37 wherein the mammal is not a human.

39. The method of statement 37 wherein the mammal is a human.

40. A method of treating cancer in a mammal comprising:

administering to the mammal a compound comprising a polypeptidecomprising a cytokine that is a ligand to ErbB-1 covalently attached tobendamustine.

41. The method of statement 40 wherein the polypeptide is a fusionprotein comprising one or more non-cytokine segments N-terminal orC-terminal to the cytokine.

42. The method of statement 40 wherein the cytokine is residues 2-54 ofSEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ IDNO:11, residues 63-148 of SEQ ID NO:12, residues 32-111 or SEQ ID NO:13,or a variant 90% or more identical to residues 2-54 of SEQ ID NO:9,residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ ID NO:11,residues 63-148 of SEQ ID NO:12, residues 32-111 or SEQ ID NO:13.43. The method of statement 40 wherein the polypeptide comprises SEQ IDNO:8.44. A compound comprising bendamustine covalently attached to apolypeptide comprising a cytokine that is a ligand to ErbB-1.

EXAMPLES

Plasmids were synthesized by DNA 2.0 (Menlo Park, Calif.) encoding theseproteins with nucleotide sequences optimized for expression in E. coli,and under the control of a T7 promoter:

Protein encoded Description Sequence 403IGF His6-IGF SEQ ID NO: 14764IGF His6-K5-IGF132 SEQ ID NO: 18 765IGF His6-K5-R3IGF SEQ ID NO: 2784IGF mutTrx-R3IGF SEQ ID NO: 15 785IGF mutTrx-IGF132 SEQ ID NO: 16E. coli BL21(DE3) was transformed with each of the plasmids andtransformants isolated. 10 ml of the transformed BL21(DE3) culture ofeach was used to seed 500 ml of LB media with 50 ug/ml kanamycin(LB-kan) in a 2 L baffled flask. These were induced with 0.4 mM finalIPTG at an O.D. 600 nm of 0.6 and grown overnight at 25 degrees C.

The cells were resuspended in 50 mM Tris-HCl pH 8.0 and frozen. Theywere thawed and incubated at 5% wet weight/volume cell weight in 50 mMTris-HCl pH 8.0, 0.2% Triton-X100, 0.5 mg lysozyme per g cell paste, for30 minutes at room temperature. They were then sonicated to break cells.MgCl₂ was added to 3 mM final concentration and 250 ul of BENZONASE wasadded per liter of culture. This was incubated a further 1 hour at roomtemperature.

Inclusion bodies were isolated by centrifugation. Soluble fraction wasretained.

Inclusion bodies were solubilized in 7 M urea, 0.5 M NaCl, 20 mMphosphate pH 7.8.

The solubilized inclusion bodies were loaded onto 1 ml ofNi-nitrolito-triacetic acid (Ni-NTA) resin in a column. The column waswashed with Ni-A buffer and eluted with Ni-B buffer.

Ni-A 6 M urea, 0.5 M NaCl, 20 mM sodium phosphate, 20 mM imidazole, pH7.3.

Ni-B 6 M urea, 0.5 M NaCl, 20 mM sodium phosphate, 0.4 M imidazole, pH7.3.

The protein yields were:

403IGF eluate 3.6 mg

764IGF eluate 16 mg

765IGF eluate 24 mg

784IGF eluate 6.7 mg

785IGF eluate 1.9 mg

SDS-PAGE was run of the eluates and of the crude insoluble and solublefractions. It appeared that 784IGF and 785IGF had about half of the IGFin the soluble fraction and half in the insoluble. 403IGF, 764IGF, and765IGF appeared to have nearly all of the IGF in the insoluble fraction.

From this data, the best yield was with 765IGF. Those with the SEQ IDNO:1 leader sequence (764IGF and 765IGF) gave better yields than thosewith a simple Met-His6 leader (403IGF) or with thioredoxin leadersequences (784IGF and 785IGF). And the constructs with the R3IGF mutantfor the IGF portion (765IGF and 784IGF) gave better yields than thecorresponding constructs with the IGF132 mutant for the IGF portion ofthe fusion protein (764IGF and 785IGF).

Example 2 Refolding and Binding Assay

2 ml of each of the original Ni eluates from Example 1 was mixed withabout an equal volume of 100 mM glycine, 6 M urea, pH 9.5, concentratedby ultrafiltration in a CENTRICON 3 kDa filter unit, then brought upagain in that buffer and concentrated to about 420 ul. Then they werediluted to 2 mg/ml for 403IGF, 764IGF, and 765IGF, and 4 mg/ml for784IGF and 2.4 mg/ml for 785IGF.

200 ul of each of these was mixed rapidly with 1.8 ml of refold buffer.Refold buffer was 1.4 M urea, 100 mM glycine, 0.5 M NaCl, 19% ethanol,0.5 mM GSSG, 4 mM GSH, pH 9.5. They were refolded at room temperaturefor 3 hours, and then tested in a binding assay for competition bindingto IGF receptors against 1-132 radioactive wild type IGF (Perkin Elmer,Inc.) For comparison, commercial Long-R3-IGF (LR3IGF) was also tested.

The approximate binding constants (KDs) in this experiment were these:

LR3IGF 1 nM 403IGF 2 nM 764IGF 100 nM  765IGF 10 nM  784IGF 3 nM 785IGF40 nM The fusion proteins containing the R3IGF mutant (LR3IGF, 765IGF, and784IGF) had lower K_(D)s than those containing the IGF132 mutant(403IGF, 764IGF and 785IGF).

Example 3 Purification and Yield of 765IGF

A plasmid encoding 765IGF with optimized codon usage for E. coli, withthe 765IGF gene under the control of a T7 promoter, was synthesized byDNA 2.0 (Menlo Park, Calif., USA). E. coli B121(DE3) was transformedwith the plasmid and grown in fermentor culture and induced with IPTG.

765IGF was purified under denaturing conditions by ion exchangechromatography and Nickel affinity chromatography. The yield of purified765IGF was about 60 mg per liter of culture.

765IGF was refolded by a procedure similar to that of Example 2 and thenthe refolded protein was purified by ion exchange chromatography.

Example 4 765IGF Binding Assay to IGF-1 Receptor

Method:

Theory of Assay:

Radioactive ¹²⁵I labeled insulin-like growth factor-1 (IGF-1) competeswith a test ligand for binding to type 1 IGF receptors that are abundanton MCF7 cells (a human breast cancer cell line) in vitro. The testedligands include our 765IGF variant of insulin-like growth factor-1(IGF-1) and our novel covalent conjugates that contain the antifolatedrug methotrexate coupled to 765IGF, as well as commercially availablelong-R3-IGF-1 (Sigma Aldrich, St. Louis, Mo., USA) as a comparison andpositive control.

MCF7 cell media: 500 mL MEM, 0.01 mg/mL bovine insulin; 5 mL sodiumpyruvate, 5 mL non-essential amino acids, 10 mL sodium bicarbonate, 10mL fetal bovine serum, 5 mL penicillin/streptomycin.

MCF7 cells (ATCC HTB-22) were plated at 20,000 cells per well in avolume of 0.5 mL/well in a 48-well tissue culture plate (flat bottomwith low evaporation lid) and placed in a cell culture incubator set at37° C. with 5% CO₂. After 2-3 days in culture the plates were washed 2×with 0.5 mL per well of cold binding assay buffer (100 mM Hepes-NaOH, pH7.2; 120 mM NaCl; 5 mM KCl, 1.2 mM MgSO4; 0.1% BSA). After the finalwash, 0.5 mL of binding assay buffer was added to each well and theplates are placed at 4° C. for 2 to 6 hours.

Test ligands were prepared at a concentration of 10 micromolar(long-R3-IGF) or 20 micromolar (765IGF and IGF-MTX) in 5 mM HCl in avolume of 200 ul. To determine the concentration, the molecular weightof 765IGF (9742 daltons) and long-R3-IGF (9111 daltons) are used. Forlong-R3, the lyophilized commercial material is dissolved at 1.0 mg/mlin 10 mM HCl and this is diluted to a concentration of 91 ug/ml for a 10uM solution.

The 765IGF and long-R3-IGF were diluted into binding buffer in the wellsat concentrations of 2000 nM to 1 nM.

Next, 25 uCi lot of I-125 IGF (Perkin Elmer Radiochemicals, Waltham,Mass., USA) was dissolved in 1 ml of water. An appropriate dilution intobinding buffer ws made, and then 50 ul of diluted radioactive IGF isadded to each well, to add 0.03 uCi or more per well. For fresh 1-125IGF, per plate used 100 ul of the 1 ml solution of I-125 IGF in watercan be added to 2.6 ml of binding buffer per plate used, and 50 ul addedper well.

The plates were then incubated overnight at 4° C. Then the liquid waswithdrawn from each well with a micropipettor and the wells were washedtwice in binding buffer. Cells were lysed with 0.5 mL 300 mM NaOH, 1%SDS and the lysates were counted on a gamma counter.

Results:

The result of an IGF-1 receptor binding assay for 765IGF andcommercially available long-R3-IGF are shown in FIG. 1. At highconcentrations, 765IGF consistently displaced more radioactivity thanlong-R3-IGF, suggesting it may bind to IGF-1 binding sites on themembranes that long-R3-IGF does not. The K_(D) of 765IGF in this assaywas less than 1 nM, while the K_(D) of long-R3-IGF was about 3 nM.

Example 5 Conjugation of Methotrexate to 765IGF

The protein was buffer exchanged into pH 7.3 conjugation buffer andadjusted to a concentration of 2.5 mg/ml.

pH 7.3 conjugation buffer: 25 mM sodium phosphate, 10 mM NaCl, 6 M urea,pH 7.3.

pH 6.3 conjugation buffer is the same buffer at pH 6.3.

Methotrexate was dissolved at 20 mg/ml in pH 6.3 conjugation buffer, andthe pH adjusted to pH 6.3 with NaOH.

1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) wasfreshly dissolved in pH 6.3 conjugation buffer at 75 mg/ml.

One volume of EDC solution was added to 1 volume of MTX solution andincubated 30 seconds at room temperature and then this mixture was addedto 8 volumes of 2.5 mg/ml protein solution in pH 7.3 conjugation buffer.

The mixture was mixed and then reacted overnight at room temperature.Then 6 M HCl was added to the reaction mixture to 60 mM finalconcentration. Then the reaction mixture was buffer exchanged into 10 mMHCl.

Result:

The amount of methotrexate conjugated per mole of protein was determinedby measuring absorbance of the conjugate at 305 nm in 100 mM HCl, usinga molar extinction coefficient for methotrexate groups of 21.6 per mM(Chamberlin et al. Analytical Profiles of Drug Substances, 1976,5:283-306.) The protein concentration was determined by quantitativeamino acid analysis. By this, the molar ratio of MTX groups to IGF inthe 765IGF-MTX conjugate was approximately 8.

Example 6 765IGF-MTX In Vitro Cytotoxicity Assay

Cytotoxicity Assay.

This potency assay is an assay for inhibition of proliferation of MCF-7tumor cells in vitro by incubation with the 765IGF-MTX.

Method

Day 0.

Five-thousand MCF7 cells were plated per well in a 96-well test plate in100 ul of rich media on day 0.

Day 1.

A shadow plate was made for each test plate, with each well of theshadow plate containing media or 3× the intended final concentration oftest agent in media in each well. As a negative control, media is used.As a positive control, free methotrexate at 3 uM is used.

After making the shadow plate, 50 ul is transferred from each well ofthe shadow plate to the corresponding well of the test plate to generatethe final concentrations of test agent in the wells of the test plate.

Day 5.

Cell proliferation is determined by adding_Dojindo CCK-8 reagent andincubating and measuring absorbance of the dye according to themanufacturer's instructions.

Result:

Results of a representative cytotoxicity assay with 765IGF-MTX are shownin FIG. 2. The IC₅₀ (Concentration needed for 50% inhibition of cellproliferation) of 765IGF-MTX was 249 nEquivalents per L. (AnanoEquivalent is a nanomole of methotrexate groups conjugated to765IGF.) For comparison, in the same assay, the IC₅₀ of freemethotrexate was measured as 88 nM.

For comparison, an LR3IGF-MTX conjugate (methotrexate conjugated tolong-R3-IGF) had an IC₅₀ of about 400 nEq/L (McTavish et al., 2009,Translational Research 153:275-282).

Example 7 Inhibition of Dihydrofolate Reductase by Methotrexate andIGF-Methotrexate Conjugates

Method:

The experiments were done with the dihydrofolate reductase assay kitfrom Sigma-Aldrich (St. Louis, Mo., USA), according to themanufacturer's instructions. In the assay dihydrofolate reductase ismixed with pH 7.5 buffer. Next the inhibitor—methotrexate or anIGF-methotrexate conjugate—is added and the solution mixed. It wasincubated for 30 seconds to allow inhibitor binding. Then NADPH is addedto 50 uM final concentration, and then dihydrofolic acid is added to 60uM final concentration. The reaction is monitored by measuringabsorbance at 340 nm.Results:

The tested conjugates were:

765IGF-MTX prepared as described in Example 4. 765IGF has 9 amino groupsavailable to conjugate to methotrexate (8 lysines and the N-terminalamino group). This batch had a MTX:protein molar ratio of 7.5.

765IGF-MTX 1/3. This conjugate was prepared with 1/3 of the usualconcentrations of MTX and EDC in the conjugation reaction. It produced aconjugate with a MTX:protein molar ratio of 1.2.

LR3IGF-MTX. In this case, the version of IGF is long-R3-IGF. This has 4available amino groups for conjugation (3 lysine side chains and theN-terminal amino group). This conjugate had a MTX:protein ratio of 2.8.

In addition, free methotrexate was tested.

The conjugates were exhaustively ultrafiltered to remove any freemethotrexate before their use in the inhibition assay.

A plot of the inhibition data for 765IGF-MTX is shown in FIG. 3.

The IC₅₀s of methotrexate and the conjugates were these:

Competitor IC₅₀ MTX:IGF ratio Methotrexate 5.3 nM N.A. 765IGF-MTX  95nEq/L 7.5 1/3 765IGF-MTX 90 1.2 LR3IGF-MTX 99 2.8The IC₅₀ in nEq/L was approximately the same for all three of theIGF-MTX conjugates, despite having different numbers of MTX groupsconjugated per IGF protein monomer. This shows that each conjugatedmethotrexate group acts as an independent inhibitor of the enzyme. Ifthe additional methotrexate groups on a conjugate monomer weresterically unable to bind to and inhibit a DHFR enzyme once one group isbound to a DHFR enzyme, then one would expect that the IC₅₀ for theconjugates would be the same in terms of nM protein concentration foreach of the conjugates, instead of being the same in terms of nEq/L MTXgroup concentration, as is observed. Because the inhibition isproportional to MTX groups, 765IGF-MTX, with its higher MTX loading, hasan inhibition constant in terms of protein concentration of 13 nM (95nEq/L divided by 7.5 MTX per IGF gives 13 nM IGF), whereas LR3IGF-MTXhas an inhibition constant in terms of protein concentration of 35 nM.Thus, with the higher loading of MTX, less 765IGF protein needs to beused to achieve the same inhibition of DHFR, and by inference the samelevel of killing of tumor cells.

The data show that the protein-conjugated MTX groups inhibit DHFR, but ahigher concentration is needed for inhibition as compared to free MTX.

Example 8 765EGF

The protein 765EGF, having SEQ ID NO:8 was synthesized:

(SEQ ID NO: 8) MVKGKHHHHHHNGKGKSK NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR.The underlined portion above is the SEQ ID NO:1 leader sequence, alsofound in 765IGF. The non-underlined portion is the amino acid sequenceof mature soluble form of human epidermal growth factor. A plasmidencoding this protein, with optimized codon usage for E. coli, under theexpression control of the T7 promoter, was synthesized by DNA 2.0 (MenloPark, Calif., USA). E. coli BL21(DE3) was transformed with the plasmidand used to express the protein. The protein was expressed with growthin 2XYT medium+2.1 g/L dextrose, 50 ug/ml kanamycin. It was induced with0.4 mM IPTG, and harvested 5 hours later.

The 765EGF protein was found in inclusion bodies. The inclusion bodieswere isolated and solubilized and the 765EGF protein was purified by ionexchange chromatography and Ni-affinity chromatography under denaturingconditions in 6 M urea and 20 mM mercaptoethanol. The yield wasexcellent: 83.5 mg purified 765EGF per liter of culture.

The Ni-purified protein at 2 mg/ml or less in the Ni elution buffer wasrefolded by slow addition with stirring to 10 volumes of refold buffer,which is 1.6 M urea, 20% 190 proof ethanol, 0.5 M NaCl, 0.1 M glycine,0.5 mM oxdiized glutathione, 4 mM reduced glutathione, pH 9.6. It wasincubated overnight at room temperature.

The refolded protein was acidified to pH 4.5 and then purified at pH 4.5by cation exchange chromatography.

The refolded protein was subjected to ESI-TOF mass spectrometry. Themass was 85% 8282, which is exactly the predicted mass for the proteinwith all of the 6 cysteines in disulfides, and 15% 8151, which is thepredicted mass of the fully oxidized protein with the initiatormethionine removed.

Example 9 765EGF-Bendamustine Conjugate

Native EGF has only two available amino groups (1 lysine side chain andthe amino terminus amine). 765EGF has 5 additional lysine side chains,giving it 7 amino groups. We attempted to conjugate to 765EGF thecarboxyl-containing cancer chemotherapy drug be bendamustine, whosestructure is shown below.

With reaction with EDC, a direct amide bond is formed between carboxylsand amino groups.

Purified and refolded 765EGF was dialyzed against pH 7.3 conjugationbuffer (6 M urea, 10 mM NaCl, 25 mM sodium phosphate, pH 7.3). Thedialyzed protein was 34 ml at 1.15 mg/ml.

Bendamustine was dissolved at 20 mg/ml in pH 6.3 conjugation buffer (5.5ml; 6 M urea, 10 mM NaCl, 25 mM sodium phosphate, pH 6.3). The pH was4.1. 180 ul of 2 M NaOH was added, which made the pH about 6.7.

EDC was dissolved 60 mg/ml immediately before use. Then 4.25 ml of 60mg/ml EDC and 4.25 ml 20 mg/ml bendamustine were mixed, and incubatedabout 30 seconds. Then this mixture was added to the 34 ml of 1.15 mg/mlEGF.

This was incubated overnight at room temperature, then pH adjusted to pH2.5, then dialyzed against 10 mM HCl. The conjugate was stored at −20degrees C.

The molecular weight of the conjugate was determined by matrix assistedlaser desorption (MALDI) mass spectrometry as a broad peak with anaverage molecular weight of 9145 (FIG. 4). The unconjugated 765EGF has amolecular weight of 8282. Each bendamustine added would add 340 massafter losing one water molecule for the conjugation, so this is 2.5bendamustine per protein. 765EGF has 7 amino groups available forconjugation (6 lysines and the amino terminus). So an average of 2.5 ofthe 7 are conjugated. The mass spectrum shows smaller amounts of massspecies out to about 10,500. The predicted mass of the species with all7 amino groups having a bendamustine conjugated would be 10,662. Themass spectrum of the conjugate has almost no specific mass peaks thatstand out, suggesting that the product has many variations and crossreactions, rather than being a simple mixture with 1, 2, 3, 4, 5, 6, or7 unmodified bendamustine groups covalently attached to otherwiseunmodified 765EGF protein.

The conjugate was also run on reducing SDS-PAGE (Data not shown). Morethan 90% of the material ran as 10 kDa monomer. Less than 10% appearedas ˜20 kDa dimer.

Example 10 Cytotoxic Activity of 765EGF-Bendamustine

A-431 human epithelial carcinoma cells were seeded at 5,000 cells perwell in 96-well plates and grown for 1 day. A-431 cells have abundantEGF receptors. After 1 day growth, free bendamustine and765EGF-bendamustine were added to the plate at concentrations rangingfrom 200 micromolar bendamstine to 6 nM and from 20 microEq/L765EGF-bendamustine down to 0.6 nM. The plates were incubated with drugat 37 degrees C. under a humidified 5% CO₂ atmosphere for 4 days, andthen DOJINDO cell counting kit-8 reagent was added to wells to readproliferation. The plates were read according to manufacturer'sinstruction. The relative proliferation of the cells with both freebendamustine and EGF-bendamustine is shown in FIG. 5. There was a hugedifference between free bendamustine and EGF-bendamustine. Freebendamustine only inhibited proliferation at concentrations above about10 micromolar and had an IC₅₀ of about 90 micromolar. EGF-bendamustinehad an IC₅₀ of 12 nanoEquilavents/L (FIG. 6). The difference ineffectiveness was more than 1000-fold expressed in terms of theconcentration of bendamustine groups.

Example 11 Tumor Xenograft Treatment In Vivo with 765EGF-Bendamustine

A-431 cells were grown and harvested, and resuspended in media. Thecells were counted, and then promptly centrifuged and resuspended in PBSat approximately 7 million cells per ml, and then mixed with an equalvolume of matrigel on ice. 100 ul containing 3.5 million cells wasinjected intradermally in the flank in each mouse. Mice were monitoredfor tumor growth. Tumor volume was calculated as (length×width²)/2. Whentumors reached 100 mm3, treatment was initiated. Mice were treated ondays 0, 7, and 14. Tumor size was measured every 3 or 4 days. Micereceived either saline only (dose 0) or EGF-bendamustine at 50, 200,800, or 3200 picoEquivalents per gram body weight by intraperitonealinjection, or 200 picoEquivalents per gram by intravenous tail veininjection. The drug was at 0.82 mg/ml protein, using a protein molecularweight of 8282 and 2.5 bendamustine groups per protein, this was 250nanoEquivalents per ml. This was diluted in 2 mM HCl, 150 mM NaCl forinjection. A volume of 300 ul was injected for IP injections and 100 ulfor IV injections.

Results:

A graph of average tumor volume for the different treatment groupsversus time is shown in FIG. 7. All of the treatment groups receiving adose of 200 pEq/g or greater had reduced tumor growth as compared to theuntreated control and the group receiving the smallest dose of 50 pEq/g.

FIG. 8 shows tumor growth (a) pooled for the groups receiving a dose of0 or 50 pEq/g, (b) pooled for the groups receiving a dose of 200 pEq/gor higher, and (c) the group receiving an IV dose of 200 pEq/g. At days14 and beyond, both the 200+ and the IV200 groups were significantlydifferent from the pooled 0+50 group (p=0.05 significance level).

Five mice were cured in the study. That is, their tumors becameundetectable and remained so to 50 days after treatment dose 1. Three often in the IV200 group were cured, and 1 of 8 receiving the IP dose of200, and 1 of 7 receiving the IP dose of 800. This is summarized inTable 1. Thus, it appeared that IV administration was more effectivethan intraperitoneal, although the difference was not statisticallysignificant.

Tumor size and tumor growth delay, was significantly different betweenmice receiving a dose of 200 pEq/g or higher versus those receiving 50pEq/g or lower, and between the mice receiving the IV 200 pEq/g dose andthe mice receiving 50 pEq/g or lower.

TABLE 1 Required more Required more than 26 days to than 34 days Never14-day average Average¹ days reach 1200 to reach 1200 reached 1200 tumorvolume to reach 1200 mm³ tumor mm³ tumor mm³ tumor (standard mm³(standard dose (pEq/g) volume volume volume error) error) 0 0/6 0/6 0/6 1048 (217) 19.7 (2.32) 50 1/6 1/6 0/6  1313 (186) 18.7 (4.73) 200 3/81/8 1/8  693 (172) 26.8 (5.52) 800 3/7 1/7 1/7  580 (124) 29.6 (3.01)3200  4/10  1/10 0/10 714 (111) 25.3 (3.28) IV 200 5/10 (a) 5/10 (e)3/10 722 (166)) (c) 36.5 (6.96) (f) 0 and 50 1/12 (a, b) 1/12 (e) 0/121180 (141) (c, d) 19.2 (2.52) (f, g) 200 or more 15/35 (b) 8/35 5/35 685(71)(d) 29.69 (2.795) (g) (¹For mice whose tumors never reached 1200mm³, a value of 62 days was used. a, b, c, d, e, f, and g: cells withthe same letter are significantly different from each other, p < 0.05).Conclusion:

765EGF-bendamustine successfully treated mouse xenografts with A-431, acell line that has EGF receptors. This cell line was almost completelyinsensitive to free bendamustine. Thus, conjugation of chemotherapyagents to EGF and EGF fusion proteins is a successful way to targettumor cells expressing EGF receptors.

Example 12 765TNFa Fusion Protein

A plasmid encoding the soluble form of tumor necrosis factor alpha(TNFa) in a fusion protein with the SEQ ID NO:1 leader sequence wassynthesized by DNA2.0 (Menlo Park, Calif., USA). The codons wereoptimized for E. coli. The coding sequence was under the expressioncontrol of a T7 promoter. The encoded protein is called 765TNFa (SEQ IDNO:17).

E. coli BL21(DE3) was transformed with the plasmid, and the transformantwas grown in LB medium with 50 ug/ml kanamycin. The culture was inducedwith 0.4 mM IPTG when it reached an O.D. 600 nm of 0.6. Culture in theamount of 400 ml was grown in each of two 2 L baffled flasks withshaking at 37° C.

Cells were harvested and broken by lysozyme treatment and sonication.Broken cells were centrifuged to remove insoluble debris. 765TNFaprotein was purified from the supernatant (soluble fraction) by Nickelaffinity chromatography. Fifty mg of purified 765TNFa was obtained from800 ml of culture. The purified 765TNFa was pure by SDS-PAGE with thepredicted mass of 19 kDa (data not shown). From a fermentor culture, 99mg of purified 765TNFa was obtained per liter of culture. These yieldsare excellent.

Activity Assay.

L929 cells were plated at 10,000 cells per well in two 96-well plates intheir recommended growth medium with 2% fetal bovine serum. They weregrown for 2 days at 37° C. in a 5% CO₂ humidified atmosphere. In oneplate, after the 2 days incubation 1 ug/ml final concentrationactinomycin D was added; in the other plate no actinomycin D was added.765TNFa was then added at a range of concentrations. Plates wereincubated 24 hours more. Then cell viability was quantified with DOJINDOcell counting kit-8 according to manufacturer's instructions. In plateswith 1 ug/ml actinomycin, the 765TNFa had an IC₅₀ for killing the cellsof less than 79 pg/ml. With no actinomycin, the IC₅₀ was about 70 ng/ml.These are even lower IC₅₀s than are reported in the literature forTNF-alpha. So 765TNFa purified as described is active, possibly moreactive than wild type TNF-alpha.

SEQUENCES

SEQ ID NO: 1 MVKGKHHHHHHNGKGKSK (765IGF) SEQ ID NO: 2MVKGKHHHHH HNGKGKSKGP RTLCGAELVD ALQFVCGDRG FYFNKPTGYG SSSRRAPQTGIVDECCFRSC DLRRLEMYCA PLKPAKSA (human IGF-1) SEQ ID NO: 3GPETLCGAEL VDALQFVCGD RGFYFNKPTG YGSSSRRAPQ TGIVDECCFR SCDLRRLEMYCAPLKPAKSA (IGF132) SEQ ID NO: 4FVNQHLCGSHLVEALYL VCGDRG FYFNKPTGYG SSSRRAPQTG IVDECCFRSCDLRRLEMYCAPLKPAKSA (long-R3-IGF) SEQ ID NO: 5MFPAMPLSSLFVN GPRTL CGALVDALQ FVCGDRGFYF NKPTGYGSSS RRAPQTGIVDECCFRSCDLR RLEMYCAPLK PAKSEA (R3-IGF) SEQ ID NO: 6GPRTLCGAELVD ALQFVCGDRG FYFNKPTGYG SSSRRAPQTGIVDECCFRSC DLRRLEMYCA PLKPAKSA (des(1-3)IGF1) SEQ ID NO: 7TLCGAELVD ALQFVCGDRG FYFNKPTGYG SSSRRAPQTGIVDECCFRSC DLRRLEMYCA PLKPAKSA (765EGF) SEQ ID NO: 8 MVKGKHHHHHHNGKGKSKNSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELRSEQ ID NO: 9, EGF precursor: (SEQ ID NO: 9)MNSDSECPLS HDGYCLHDGV CMYIEALDKY ACNCVVGYIG ERCQYRDLKW WELRSEQ ID NO: 10, TGFα precursor (SEQ ID NO: 10)MVPSAGQLAL FALGIVLAAC QALENSTSPL SADPPVAAAV VSHFNDCPDSHTQFCFHGTC RFLVQEDKPA CVCHSGYVGA RCEHADLLAV VAASQKKQAITALVVVSIVA LAVLIITCVL IHCCQVRKHC EWCRALICRH EKPSALLKGR TACCHSETVVSEQ ID NO: 11, Amphiregulin precursor: (SEQ ID NO: 11)MRAPLLPPAP VVLSLLILGS GHYAAGLDLN DTYSGKREPF SGDHSADGFEVTSRSEMSSG SEISPVSEMP SSSEPSSGAD YDYSEEYDNE PQIPGYIVDDSVRVEQVVKP PQNKTESENT SDKPKRKKKG GKNGKNRRNR KKKNPCNAEFQNFCIHGECK YIEHLEAVTC KCQQEYFGER CGEKSMKTHS MIDSSLSKIALAAIAAFMSA VILTAVAVIT VQLRRQYVRK YEGEAEERKK LRQENGNVHA IASEQ ID NO: 12, HD-EGF precursor (SEQ ID NO: 12)MKLLPSVVLK LLLAAVLSAL VTGESLEQLR RGLAAGTSNP DPSTGSTDQLLRLGGGRDRK VRDLQEADLD LLRVTLSSKP QALATPSKEE HGKRKKKGKGLGKKRDPCLR KYKDFCIHGE CKYVKELRAP SCICHPGYHG ERCHGLSLPVENRLYTYDHT TILAVVAVVL SSVCLLVIVG LLMFRYHRRG GYDVENEEKV KLGMTNSHSEQ ID NO: 13, Betacellulin precursor: (SEQ ID NO: 13)MDRAARCSGA SSLPLLLALA LGLVILHCVV ADGNSTRSPE TNGLLCGDPEENCAATTTQS KRKGHFSRCP KQYKHYCIKG RCRFVVAEQT PSCVCDEGYIGARCERVDLF YLRGDRGQIL VICLIAVMVV FIILVIGVCT CCHPLRKRRKRKKKEEEMET LGKDITPINE DIEETNIA  403IGF SEQ ID NO: 14 MTSGHHHHHHSAGVNG FVNQHLCGSHL VEALYLVCGD RGFYFNKPTG YGSSSRRAPQTGIVDECCFR SCDLRRLEMY CAPLKPAKSA, 784IGF SEQ ID NO: 15 MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGHCKMIKPFFHSLSEKYSNVIFLEVDVDDSQDVASESEVKSMPTFQFFKKGQKVGEFSGANKEKLEATINELVGSKSGHHHHHHSAKGGPRTLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA, 785IGF SEQ ID NO: 16 MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGHCKMIKPFFHSLSEKYSNVIFLEVDVDDSQDVASESEVKSMPTFQFFKKGQKVGEFSGANKEKLEATINELVGSKSGHHHHHHSAKGFVNQHLCGSHLVEALYLVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA, 765TNFa SEQ ID NO: 17  MVKGKHHHHHHNGKGKSKVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELRDNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLLSAIKSPCQRE TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL,764IGF SEQ ID NO: 18 MVKGKHHHHHHNGKGKSKFVNQHLCGSHLVEALYLVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA,

All patents, patent documents, and other references cited areincorporated by reference.

What is claimed is:
 1. A fusion protein comprising either SEQ ID NO:1 orresidues 2-18 of SEQ ID NO:1; fused directly to the N-terminus of acytokine.
 2. The fusion protein of claim 1 wherein the cytokine is aligand to ErbB-1 or IGFR1.
 3. The fusion protein of claim 1 wherein thecytokine is tumor necrosis factor-alpha.
 4. The fusion protein of claim3 comprising SEQ ID NO: 17 or residues 2-175 of SEQ ID NO:
 17. 5. Thefusion protein of claim 1 wherein the cytokine is selected from a groupconsisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, and residues32-111 of SEQ ID NO:13.
 6. The fusion protein of claim 5 wherein thecytokine is SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:7. 7.The fusion protein of claim 5 wherein the cytokine is residues 2-54 ofSEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues 101-184 of SEQ IDNO:11, residues 63-148 of SEQ ID NO:12, or residues 32-111 of SEQ IDNO:13.
 8. The fusion protein of claim 5 wherein the fusion proteincomprises SEQ ID NO:2 or residues 2-88 of SEQ ID NO:2.
 9. The fusionprotein of claim 5 wherein the cytokine is residues 2-54 of SEQ ID NO:9.10. A fusion protein consisting of either SEQ ID NO:1 or residues 2-18of SEQ ID NO:1 fused to the N terminus of a cytokine selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,residues 2-54 of SEQ ID NO:9, residues 40-89 of SEQ ID NO:10, residues101-184 of SEQ ID NO:11, residues 63-148 of SEQ ID NO:12, or residues32-111 of SEQ ID NO:13.
 11. The fusion protein of claim 10 consisting ofSEQ ID NO:2 or residues 2-88 of SEQ ID NO:2.